Molecular analyses of primary afferent nociceptors have revealed remarkable heterogeneity. In addition to the traditional peptide and non-peptide classes of nociceptor, there is a complex array of thermal, mechanical and chemical transducers that define pain fibers. Our laboratory research is focused on the nervous system mechanisms that underlie the development of persistent pain. Among the important questions are whether and the extent to which neurochemically-distinct nociceptor classes target different populations of spinal cord projection neurons, the extent to which different nociresponsive projection neurons engage different brain regions and the extent to which local (interneuronal) and descending circuits that influence one population of projection neuron are recapitulated for other spinal cord nociresponsive networks. Unfortunately, traditional neuroanatomical tract tracing methods cannot provide the answers to these questions. To overcome these limitations, we have developed powerful genetically-based transneuronal anterograde tracing methods to dissect the circuits engaged by distinct populations of nociceptor. The experiments outlined in Specific Aim 1 will continue these studies, focusing on the peptide, non- peptide and TRPV1 subsets of nociceptors. Using a very novel tracing method described in Specific Aim 2, we will also examine the circuits that influence the major populations of spinal cord projection neuron. We will use Cre-recombinase-dependent pseudorabies virus transneuronal retrograde tracing to study selectively the circuits that regulate different populations of spinal cord projection neuron. Finally, experiments in Specific Aim 3 will address the circuits that regulate the protein kinase C gamma subset of spinal cord interneurons, which we previously implicated in the generation of injury- induced persistent pain. Together our studies will not only provide information on the central nervous system circuits engaged by subsets of nociceptors, but will define important differences in the circuits that regulate what are clearly heterogeneous populations of nociresponsive spinal cord projection neurons. The information gained from these studies will contribute to the development of targeted pain therapies that are based on interrupting specific circuits that underlie particular pain conditions. PUBLIC HEALTH RELEVANCE Our laboratory has identified many of the molecular properties that underlie the incredible heterogeneity of the peripheral nerve fibers that respond to injury, which is the first step in the process through which tissue or nerve injury produces acute and, in pathological conditions, chronic pain. With a view to defining the neural circuits through which the information from the pain fibers is communicated to the spinal cord and brain, where the pain percept is eventually generated, we have developed powerful genetically-based neuroanatomical tracing methods that we will exploit in the proposed series of experiments. The information gained from these studies will contribute to the development of targeted pain therapies that are based on interrupting specific circuits that underlie particular pain conditions.